Principle:

Viruses can grow only in living systems. They cannot grow in non-living media like nutrient agar or nutrient broth. Therefore, their cultivation needs host cells susceptible to the specific virus.

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The virus such as bacteriophage, which infects and grows in bacteria cells are cultivated using cultures of bacteria cells as the living system.

For example, the virus coliphage (a bacteriophage) is cultivated using the culture of the bacteria E. coli. In contrast, animal viruses, which infect and grow in the body of animals, require susceptible living animal systems.

The three living animal systems used for cultivation of animal viruses in the laboratories are as follows:

1. Susceptible Animals:

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In this technique, the virus to be cultivated is allowed to grow in the body of a living animal such as mouse or guinea pig, which is susceptible to that virus. This technique is no longer used having been replaced by the following more recent, efficient and economic techniques.

2. Tissue Culture:

It is a highly sophisticated technique. Here animal cells known to be susceptible to the virus to be cultivated and also known to multiply rapidly are first isolated from suitable living tissue of an animal. These cells are placed in a glass or plastic vessel containing an extremely rich nutritional medium. The cells attach to the surface of the vessel and continue dividing until the entire surface is covered with a confluent monolayer of cells. This is called tissue culture.

Then, the virus to be cultivated is inoculated into the susceptible tissue culture. The virus infects the living cells of the tissue culture, subvert their metabolic machinery and replicate rapidly, thereby growing profusely in the cells.

3. Embryonated Chick Eggs:

In this simple and economic technique, the virus to be cultivated is injected into an embryonated chick egg. The virus grows within the living chick egg and causes disease in the embryo, manifested by several disease symptoms (cytopathogenic effects) specific to that virus. Presence of these symptoms indicates the growth of that virus within the egg.

Procedure:

1. Two embryonated chick eggs are candled using a candling device to demonstrate the viability of the embryo. The embryo is viable, if it shows movement in response to heat from light. Also the positions of the air sac and large blood vessels are determined and marked on the egg shell during candling (Figure 8.7).

2. The shell is disinfected over the air sac with tincture iodine and then allowed to dry. The same place is swabbed with absorbent cotton soaked with 70% alcohol.

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3. The tip of a small drill is sterilized by dipping in 70% alcohol and then flaming it.

4. Using this drill, a small hole is made aseptically on the shell over the air sac in a region away from the blood vessels.

5. 0.2 ml of the 1:2 dilution of Newcastle virus is aseptically injected into the allantoic cavity of one of the two eggs using a sterilized syringe. This is done by holding the egg in a vertical position, inserting the needle of the syringe through the hole on the shell to its hilt at a 45° angle, piercing the air sac membrane and penetrating into the allantoic cavity. This virus- inoculated egg serves as the ‘test egg’.

6. After inoculation, the needle is withdrawn and the hole on the shell is sealed with sterile hot vasper.

7. Using the same technique, 0.2 ml of sterile saline is inoculated into the other embryonated chick egg, which serves as the ‘control egg’.

8. Both the eggs are incubated at 37°C for 3 to 4 days in an incubator with proper humidity.

9. The observations are noted every day.

10. Once death has been determined, the embryo is dislodged from its shell and the contents poured into a petri dish and again observed.

11. All contaminated materials are discarded into a beaker containing bleaching powder.

Observations:

1. The eggs are candled everyday during incubation and observed for embryonic death as evidenced by cessation of movement, which usually occurs 3 to 4 days following inoculation of the virus.

2. Once death has been determined, the embryo is dislodged from its shell and the contents poured into a petri dish. It is observed for necrotic lesions and evidence of hemorrhage.

3. The control egg is examined for evidence of cytopathogenic effects.